The present invention relates to cancer therapy and to novel anticancer agents having a mechanism of action which is quite specific. It also relates to novel chemical compounds as well as their therapeutic application in humans.
The present invention relates to the use of novel non-nucleotide chemical compounds which interact with specific structures of deoxyribonucleic acid (DNA). These novel compounds consist of a distribution agent linked to two aminoaromatic groups. These novel compounds are useful in the treatment of cancers and act in particular as telomerase-inhibiting agents. They are particularly useful for stabilizing DNA in G-quadruplex structure (guanine tetrads). The therapeutic application of the inhibition of telomerase via the stabilization of these G-quadruplexes is the termination of cellular mitosis and the death of rapidly dividing cells such as cancer cells and possibly the induction of the senescence of cancer cells.
The compounds of the present invention have the advantage, from the therapeutic point of view, of blocking telomerase. From a biological point of view, telomerase allows the addition of repetitive DNA sequences of the TTAGGG type, termed telomeric sequences, at the end of the telomer, during cell division. Through this action, telomerase renders the cell immortal. Indeed, in the absence of this enzymatic activity, the cell loses, at each division, 100 to 150 bases, which rapidly renders it senescent. During the appearance of rapidly dividing cancer cells, it appeared that these cells possessed telomers which were maintained at a stable length during cell division. In these cancer cells, it appeared that telomerase was highly activated and that it allowed the addition of repetitive motifs of telomeric sequences at the end of the telomer and therefore allowed conservation of the length of the telomer in the cancer cells. It appeared for some time that more than 85% of cancer cells showed positive tests for the presence of telomerase whereas somatic cells do not show this characteristic.
Thus, telomerase is a highly coveted target for treating cancer cells. The first obvious approach for blocking telomerase was the use of nucleotide structures (Chen et al., Proc. Natl. Acad. Sci. USA 93(7), 2635-2639). Among the non-nucleotide compounds which have been used in the prior art, there may be mentioned the diaminoanthraquinones (Sun et al., J. Med. Chem. 40(14), 2113-6) or the diethyloxadicarbocyanins (Wheelhouse R. T. et al., J. Am. Chem. Soc. 1998(120), 3261-2).
Patent WO 99/40087 describes the use of compounds which interact with the G-quadruplex structures which are perylene compounds and carbocyanins containing at least seven rings including two heterocycles.
It appeared, quite surprisingly, that simple structures made it possible to obtain a result which is at least equivalent with structures which are a lot less complicated from a chemical point of view. The compounds of the present invention which meet the intended objective, that is to say which bind the G-quadruplex structure and thereby exhibit a telomerase-inhibiting activity, correspond to the following general formula:
nitrogen-containing aromatic ringxe2x80x94NR3xe2x80x94distribution agentxe2x80x94NRxe2x80x23xe2x80x94aromatic ring
in which
the nitrogen-containing aromatic ring represents:
a quinoline optionally substituted with at least one group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain C1-C4 alkyl and/or alkoxy radical and/or
a quinoline possessing a nitrogen atom in quaternary form or
a benzamidine or
a pyridine
the aromatic ring represents
a quinoline optionally substituted with at least one group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain C1-C4 alkyl and/or alkoxy radical and/or
a quinoline possessing a nitrogen atom in quaternary form or
a benzamidine or
a pyridine or
a phenyl ring optionally substituted at the meta or para position with a halogen group, C1-C4 alkoxy group, cyano group, carbonylamino group optionally substituted with one or more C1-C4 alkyl groups, guanyl groups, C1-C4 alkylthio groups, amino groups, C1-C4 alkylamino groups, C1-C4 dialkylamino groups for each alkyl group, nitro group, alkylene-amino group or alkenyleneamino group or
a mono- or bi- or tricyclic hetero-cyclic ring comprising 0 to 2 heteroatoms per ring provided that at least one heteroatom is present in at least one ring optionally substituted with one or more C1-C4 alkyl groups or with alkylene or alkenylene groups
R3 and Rxe2x80x23, which are identical or different, represent independently of one another hydrogen or a C1-C4 alkyl radical
the distribution agent represents:
a triazine group optionally substituted with an alkyl radical having 1 to 4 carbon atoms, a thio, oxy or amino radical which are themselves optionally substituted with one or more short-chain alkyl chains containing 1 to 4 carbon atoms or a halogen atom or
a carbonyl group or
a group C(xe2x95x90NH)xe2x80x94NHxe2x80x94C(xe2x95x90NH) or
an alkyldiyl group containing 3 to 7 carbon atoms or
a diazine group optionally substituted with the same groups as the triazine
or one of its salts.
For the purposes of the above formula, nitrogen-containing aromatic ring is understood to mean a heterocycle comprising at least one nitrogen atom or an aromatic group containing no heteroatom in the ring but containing at least one nitrogen atom in a hydrocarbon chain attached to the ring, such as for example a guanidino or guanyl chain.
Among all the compounds included above, the use of those comprising, as distribution agent, a triazine or diazine group is preferred. Among the diazine groups, the use of pyrimidines is preferred. Among the triazines, those preferred are the compounds corresponding to formula (I) below: 
in which:
A represents
an amino group of formula NR1R2 in which R1 and R2, which are identical or different, represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or
a group OR1 or SR1 in which R1 has the same meaning as above or
an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or
a hydrogen atom or
a halogen atom chosen from fluorine, chlorine, bromine or iodine
R3 and Rxe2x80x23, which are identical or different, represent independently of one another hydrogen or a C1-C4 alkyl radical
Ar1 and Ar2, which are identical or different, represent
1. when Ar, and Ar2 are identical:
a quinoline motif optionally substituted with at least one group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl and/or alkoxy radical containing 1 to 4 carbon atoms or
a quinoline possessing a nitrogen atom in quaternary form or
a benzamidine or
a pyridine attached at the 4-position or fused with an aryl or heteroaryl group optionally substituted with a C1-C4 alkyl group
2. when Ar1 and Ar2 are different
Ar1 and Ar2 both represent one of the possibilities mentioned above for Ar1 and Ar2 or
Ar1 represents one of the above possibilities and Ar2 represents
a phenyl ring optionally substituted at the meta or para position with a halogen group, C1-C4 alkoxy group, cyano group, carbonylamino group optionally substituted with one or more C1-C4 alkyl groups, guanyl groups, C1-C4 alkylthio groups, amino groups, C1-C4 alkylamino groups, C1-C4 dialkylamino groups for each alkyl group, nitro group, alkyleneamino group or alkenyleneamino group
a mono- or bi- or tricyclic hetero-cyclic ring comprising 0 to 2 heteroatoms per ring provided that at least one heteroatom is present in at least one ring optionally substituted with one or more C1-C4 alkyl groups or with alkylene or alkenylene groups
or one of its salts.
It is evident that the quinoline motifs may be substituted by any other group not involved in the intended application; thus, acridine or isoquinoline or quinazoline or quinoxaline or phthalazine or benzothiazine or benzoxazine or phenoxazine or phenothiazine groups are included in the definition of the quinoline groups.
Among the above compounds of formula (I), there are preferred those comprising two heterocycles chosen from the 4-aminoquinolyl, 4-aminoquinolinium or quinolinium groups in which the quinolinium ring is optionally substituted with a methyl group.
As regards the R1 and R2 groups, they preferably represent the methylthio, amino, alkylamino or dialkylamino radical, in which radicals the alkyl groups possess 1 to 4 carbon atoms.
The following compounds may be mentioned by way of representative compounds of formula (I):
2-amino-bis-4,6-[(1xe2x80x2-methyl-4xe2x80x2-amino-6xe2x80x2-quinaldinio)amino]triazine dichloride
2-amino-bis-4,6-[(1xe2x80x2-ethyl-4xe2x80x2-amino-6xe2x80x2-quinaldinio)amino]triazine dichloride
2-dimethylamino-bis-4,6-[(1xe2x80x2-methyl-4xe2x80x2-amino-6xe2x80x2-quinaldinio)amino]triazine dichloride
2-methylamino-bis-4,6-[(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)amino]triazine trihydrochloride
2-amino-bis-4,6-[(1xe2x80x2-methyl-6xe2x80x2-quinolinio)-amino]triazine dichloride
2-methylamino-bis-4,6-[(4xe2x80x2-methylamino-6xe2x80x2-quinaldinyl)amino]triazine dichloride trihydrochloride
2-amino-bis-4,6-[(9xe2x80x2-amino-10xe2x80x2-methyl-2xe2x80x2-acridinio)amino)triazine dichloride hydrochloride
2-amino-bis-4,6-[(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)-amino]triazine trihydrochloride
2-amino-bis-4,6-(p-amidinoanilino)triazine trihydrochloride
2-methylthio-bis-4,6-[(1xe2x80x2-methyl-4xe2x80x2-amino-6xe2x80x2-quinaldinio)amino]triazine dichloride
2-chloro-bis-4,6-[(4xe2x80x2-dimethylamino-6xe2x80x2-quinaldinyl)amino]triazine dihydrochloride dihydrate
2-methylthio-bis-4,6-[(4xe2x80x2-dimethylamino-6xe2x80x2-quinaldinyl)amino]triazine hydrate
N,Nxe2x80x2-(4-amino-6-quinaldinyl)urea dihydrochloride
N1,N5-bis(7-chloro-1-methyl-4-quinolinio)-pentane-1,5-diamine diiodide
bis-2,4-[(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)amino]-pyrimidine trihydrochloride pentahydrate
1,5-(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)biguanide trihydrochloride dihydrate
6-[4-(4-amino-2-methylquinolin-6-ylamino)-6-methylsulphanyl-[1,3,5]triazin-2-ylamino]-2-methyl-quinolin-4-ol
N6-[4-(4-dimethylamino-2-methylquinolin-6-ylamino)-6-methylsulphanyl-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamine
N6-[4-(4-amino-2-methylquinolin-6-ylamino)-6-methylsulphanyl-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamine
N6-[4-(4-methoxy-2-methylquinolin-6-ylamino)-6-methylsulphanyl-[1,3,5]triazin-2-yl]-4-methoxy-2-methylquinolin-6-amine
Another subject of the present invention relates to the compounds of formula (I) as novel chemical products. It therefore relates to the novel products corresponding to the following formula (I): 
in which:
A represents
an amino group of formula NR1R2 in which R1 and R2, which are identical or different, represent a straight or branched alkyl group containing 1 to 4 carbon atoms or
a group OR1 or SR1 in which R1 represents hydrogen or has the same meaning as above or
an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or
a hydrogen atom or
a halogen atom chosen from fluorine, chlorine, bromine or iodine
R3 and Rxe2x80x23, which are identical or different, represent independently of one another a hydrogen atom or a C1-C4 alkyl group
Ar1 and Ar2, which are identical or different, represent
1. when Ar, and Ar2 are identical:
a quinoline motif optionally substituted with at least one group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl and/or alkoxy radical containing 1 to 4 carbon atoms and/or
a quinoline possessing a nitrogen atom in quaternary form or
a benzamidine except in the case where A represents diethylamine, hydrogen or an amine group
a pyridine attached at the 4-position or fused with an aryl or heteroaryl group optionally substituted with a C1-C4 alkyl group
2. when Ar1 and Ar2 are different
Ar1 and Ar2 both represent one of the possibilities mentioned above for Ar1 and Ar2 or
Ar1 represents one of the above possibilities and Ar2 represents
a phenyl ring optionally substituted at the meta or para position with a halogen group, C1-C4 alkoxy group, cyano group, carbonylamino group optionally substituted with one or more C1-C4 alkyl groups, guanyl groups, C1-C4 alkylthio groups, amino groups, C1-C4 alkylamino groups, C1-C4 dialkylamino groups for each alkyl group, nitro group, alkyleneamino group or alkenyleneamino group
a mono- or bi- or tricyclic hetero-cyclic ring comprising 0 to 2 heteroatoms per ring provided that at least one heteroatom is present in at least one ring optionally substituted with one or more C1-C4 alkyl groups or with alkylene or alkenylene groups
or one of its salts excluding 2-amino-bis-4,6-[(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)amino]triazine dihydrochloride and 2-amino-bis-4,6-(p-amidinoanilino)triazine dihydrochloride.
Indeed, the first of these two compounds is described in a publication which appeared under the reference Indian Journal of Animal Sciences 43 (4), pages 226-29, as antitrypanosome agent for animals and in no case as antitelomerase agent and the second compound is also described as antitrypanosome agent in J. Chem. Soc., 1960, 4525.
The compounds of formula (I) which are preferred are those for which Ar1 and Ar2 represent a group chosen from the following motifs: 4-amino- or 4-methylamino- or 4-dimethylamino-quinolyl or quinolinium in which the quinolinium ring is optionally substituted with a methyl group.
The compounds of general formula (I) which are preferred are those for which A represents an amino or dimethylamino or, more preferably, methylthio group.
There are most particularly preferred the compounds of formula (I) for which when Ar1 and Ar2 are different:
1. Ar1 represents:
a quinoline motif substituted with at least one group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl or alkoxy radical containing 1 to 4 carbon atoms and/or
a quinoline possessing a nitrogen atom in quaternary form or
a benzamidine except in the case where A represents diethylamine, hydrogen or an amine group or
a pyridine attached at the 4-position or fused with an aryl or heteroaryl group
2. Ar2 represents
a ring as defined above but different or
a phenyl ring optionally substituted at the meta or para position with a halogen, methoxy, cyano, carbonyl-amino, guanyl, methylthio, amino, methylamino, dimethylamino, morpholine, alkyleneamino or alkenyleneamino group
a quinoline, benzimidazole, indole, benzothiophene, benzofuran, benzothiazol, benzoxazol, carbazol, quinazoline or quinoxaline ring optionally substituted with one or more C1-C4 alkyl groups or with alkylene or alkenylene groups
or one of its salts excluding 2-amino-bis-4,6-[(4xe2x80x2-amino-6xe2x80x2-quinaldinyl)amino]triazine dihydro-chloride and 2-amino-bis-4,6-(p-amidinoanilino)-triazine.
Another subject of the present invention relates to the use of the compounds of formula (I) as pharmaceutical product for human use.
The methods of preparing the compounds of formula (I) 
are described below.
According to a first preparation method, compounds of general formula (I) in which Ar1 and Ar2 are identical and defined as above and R represents a halogen atom such as chlorine or fluorine, an amino, alkylamino or dialkylamino function in which the straight or branched alkyl portions contain from 1 to 4 carbon atoms, an alkyloxy or alkylthio function in which the straight or branched alkyl portions contain from 1 to 4 carbon atoms, may be obtained by amination of a dihalotriazine, most generally a dichloro-s-triazine, of general formula (B) in which A is as defined above, with an aromatic or heteroaromatic amine of general formula (C) in which Ar is as defined above, the procedure being carried out according to scheme 1: 
In the case where A represents a halogen atom, it is useful to react the corresponding 2,4,6-trihalo-s-triazine of general formula (B) with the aromatic or heteroaromatic amine ArNH2 of general formula (C).
The procedure is generally carried out by condensing one mole of dihalo-s-triazine, or trihalo-s-triazine, with 2 moles of aromatic or heteroaromatic amine. The reaction takes place in an inert medium under the reaction conditions. There may be mentioned, among the inert solvents, acetone which is optionally aqueous or an alcohol which is optionally aqueous such as ethanol, or a halogenated solvent such as dichloromethane, or an ether such as diethyl ether or dioxane, or a polar aprotic solvent such as DMF, DMSO or NMP. The procedure is preferably carried out at a temperature of between 20xc2x0 C. and the reflux temperature, in the presence in particular of an organic base such as triethylamine, or an inorganic base such as sodium hydroxide or sodium or potassium carbonate. It is also possible not to use a base during the amination reaction, and to isolate a hydrochloride of the product of general formula (A), whose base can then be released.
The dihalo- or trihalo-s-triazines of general formula (B) are either commercially available or are known, and may be obtained under the conditions described in the literature.
The aromatic or heteroaromatic amines of general formula (C) are either known or may be easily prepared by the known methods of synthesizing aromatic or heteroaromatic amines.
In the case where Ar1 and Ar2 are different, the triazine of general formula (A) may be obtained by sequential displacement of the halogen atoms, most generally of the chlorine atoms, from the products of general formula (B) by the amines Ar1 and then Ar2 of general formula (C) according to scheme 2: 
Generally, the procedure is carried out with 1 mole of dihalo-s-triazine, or trihalo-s-triazine, and 1 mole of amine Ar1. The procedure is preferably carried out in an inert solvent such as acetone which is optionally aqueous or an alcohol which is optionally aqueous, such as ethanol, or a halogenated solvent such as dichloromethane, or an ether such as diethyl ether or dioxane, or a polar aprotic solvent such as DMF, DMSO or NMP. According to a better way of carrying out the invention, the procedure is carried out at a temperature of between 20xc2x0 C. and 50xc2x0 C. Next, 1 mole of amine Ar2 is added to the product of general formula (D), which may be optionally isolated. The procedure is carried out in particular at a temperature of between 50xc2x0 C. and the reflux temperature.
Advantageously, it is possible to carry out the procedure under the conditions described in J. Fluor. Chem., 1988, 39(1), 117-123.
According to a second method, the products of general formula (A) in which Ar are as defined above and R represents a group NR1R2 or OR1 or SR1 may also be prepared by nucleophilic displacement of a halogen atom, generally a chlorine atom, from a product of general formula (A) in which R represents a halogen atom according to scheme 3: 
The procedure is generally carried out by condensing 1 mole of product of general formula (A) in which R represents a halogen atom, preferably a chlorine atom, with 1 mole of amine R1R2NH or alcoholate R10xe2x88x92 or thioalcoholate R1S. The reaction takes place in an inert medium under the reaction conditions. There may be mentioned among the inert solvents acetone which is optionally aqueous or an alcohol which is optionally aqueous such as ethanol, or a halogenated solvent such as dichloromethane, or an ether such as diethyl ether or dioxane, or a polar aprotic solvent such as DMF, DMSO or NMP. When the entering group represents a group R1R2NH, the procedure is preferably carried out at a temperature of between 20xc2x0 C. and the reflux temperature, in the presence in particular of an organic base such as triethylamine, or an inorganic base such as sodium hydroxide or sodium or potassium carbonate. It is also possible not to use a base during the amination reaction, and to isolate a hydrochloride of the product of general formula (A), the base of which can then be released. When the entering group represents a group R10xe2x88x92 or R1Sxe2x88x92, the procedure is preferably carried out with an alkali metal or alkaline-earth metal alcoholate or thioalcoholate, such as a sodium or potassium or lithium or ammonium or caesium or barium salt, in a polar aprotic solvent such as DMF or DMSO or NMP, at a temperature of between 50xc2x0 C. and the reflux temperature.
According to a third preparation method, the compounds for which R represents a hydrogen atom or a straight or branched alkyl group containing from 1 to 4 carbon atoms may also be prepared by condensation of a bisguanide of general formula (E), in which Ar1 and Ar2 are identical or different, with an acid derivative, preferably an acid chloride or a methyl ester of general formula (F) according to scheme 4: 
The condensation between the bisguanide of general formula (E) and the acid derivative of general formula (F) is generally carried out in an alcohol such as methanol or ethanol. The procedure is preferably carried out at a temperature of between 0xc2x0 C. and the reflux temperature.
The symmetric or asymmetric bisguanides of general formula (E) may be obtained by carrying out the procedure under the conditions described in the literature and in particular according to Patent J.P. 94-4993.
The products of general formula (A), in which Ar1 and Ar2 are identical, as defined above and represented by Ar, and where R represents a straight or branched alkyl group containing from 1 to 4 carbon atoms, may also be prepared by condensation of a cyanoguanidine of general formula (G), in which Ar is as defined above, with a nitrile of general formula (H) according to scheme 4: 
The condensation of the cyanoguanidine of general formula (G) with the nitrile of general formula (H) is in particular performed by carrying out the procedure at the reflux temperature of a polar solvent with a high boiling point such as 2-methoxyethanol or 1,2-dimethoxyethane.
The cyanoguanidines of general formula (G) may be prepared under the conditions described in the literature.
It is understood that the s-triazines of general formula may be obtained in the form of libraries, by applying the methods described in schemes 1, 2, 3, 4 or 5 in parallel and/or combinatorial chemistry in liquid phase or in solid phase, it being understood that when the work is carried out in solid phase, any of the reagents is attached beforehand onto a solid support, chosen according to the chemical reaction involved, and that said chemical reaction is followed by an operation of cleaving the product of the reaction from the solid support.
The present invention also relates to therapeutic compositions containing a compound according to the invention, in combination with a pharmaceutically acceptable carrier according to the mode of administration chosen. The pharmaceutical composition may be provided in solid, liquid or liposome form.
Among the solid compositions, there may be mentioned powders, gelatin capsules and tablets. Among the oral forms, it is also possible to include the solid forms which are protected from the acidic medium of the stomach. The carriers used for the solid forms consist in particular of inorganic carriers such as phosphates, carbonates or organic carriers such as lactose, celluloses, starch or polymers. The liquid forms consist of solutions, suspensions or dispersions. They contain, as dispersive carrier, either water or an organic solvent (ethanol, NMP and the like) or mixtures of surfactants and solvents or of complexing agents and solvents.
The administered dose of the compounds of the invention will be adjusted by the practitioner according to the route of administration, the patient and the condition of the latter.
The compounds of the present invention may be administered alone or mixed with other anticancer agents. Among the possible combinations, there may be mentioned
alkylating agents and in particular cyclophosphamide, melphalan, ifosfamide, chlorambucil, busulfan, thiotepa, prednimustine, carmustine, lomustine, semustine, streptozotocin, decarbazine, temozolomide, procarbazine and hexamethylmelamine
platinum derivatives such as in particular cisplatin, carboplatin or oxaliplatin
antibiotic agents such as in particular bleomycin, mitomycin, dactinomycin,
antimicrotubule agents such as in particular vinblastine, vincristine, vindesine, vinorelbine, taxoids (paclitaxel and docetaxel)
anthracyclines such as in particular doxorubicin, daunorubicin, idarubicin, epirubicin, mitoxantrone, losoxantrone
group I and II topoisomerases such as etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex,
fluoropyrimidines such as 5-fluorouracil, UFT, floxuridine,
cytidine analogues such as 5-azacytidine, cytarabine, gemcitabine, 6-mercaptomurine, 6-thioguanine
adenosine analogues such as pentostatin, cytarabine or fludarabine phosphate
methotrexate and folinic acid
various enzymes and compounds such as L-asparaginase, hydroxyurea, trans-retinoic acid, suramine, dexrazoxane, amifostine, herceptin as well as oestrogenic and androgenic hormones.
It is also possible to combine a radiation treatment with the compounds of the present invention. This treatment may be administered simultaneously, separately or sequentially. The treatment will be adapted to the patient to be treated by the practitioner.
The G-quadruplex stabilizing activity may be determined by a method using the formation of a complex with fluorescein of which the experimental protocol is described below.
All the nucleotides, modified or otherwise, were synthesized by Eurogentec SA, Seraing, Belgium. The oligonucleotide FAM+DABCYL carries the catalogue reference OL-0371-0802. It has the sequence: GGGTTAGGGTTAGGGTTAGGG corresponding to 3.5 repeats of the human telomeric motif (strand rich in G). The fluorescein is attached to the 5xe2x80x2 end, the DABCYL to the 3xe2x80x2 end, by the chemical arms described by Eurogentec. The concentration of the samples is checked by spectrophotometry, recording the absorbance spectrum between 220 and 700 nm and using the molar extinction coefficient provided by the supplier.
All the experiments were carried out in a 10 mM sodium cacodylate buffer pH 7.6 containing 0.1 M lithium chloride (or sodium chloride). The absence of fluorescent contamination in the buffer was checked beforehand. The fluorescent oligonucleotide is added at the final concentration of 0.2 xcexcM.
All the measurements of fluorescence were carried out on a Spex Fluorolog DM1B apparatus, using an excitation line width of 1.8 nm and an emission line width of 4.5 nm. The samples are placed in a microquartz cuvette of 0.2xc3x971 cm. The temperature of the sample is controlled by an external water bath. The oligonucleotide alone was analysed at 20, 30, 40, 50, 60, 70 and 80xc2x0 C. The emission spectra are recorded using an excitation wavelength of 470 nm. The excitation spectra are recorded using either 515 nm or 588 nm as emission wavelength. The spectra are corrected for the response of the instrument by reference curves. A high extinction (80-90%) of the fluorescence of fluorescein at room temperature is observed, in agreement with an intramolecular folding of the oligonucleotide at 20xc2x0 C. in the form of a G-quadruplex, which induces juxtaposition of its 5xe2x80x2 and 3xe2x80x2 ends which are respectively linked to fluorescein and to DABCYL. This juxtaposition causes an already-described phenomenon of extinction of fluorescence which is used for xe2x80x9cmolecular beaconsxe2x80x9d.
An oligonucleotide stock solution at the strand concentration of 0.2 xcexcM in 0.1 M LiCl, 10 mM cacodylate buffer, pH 7.6, is prepared beforehand, heated briefly at 90xc2x0 C. and slowly cooled to 20xc2x0 C., and then distributed in aliquots of 600 xcexcl in the fluorescence cuvettes. 3 xcexcl of water (for the control) or 3 xcexcl of test product (stock at 200 xcexcM, final concentration 1 xcexcM) are then added and mixed. The samples are then allowed to incubate for at least 1 hour at 20xc2x0 C. before each measurement. The use of longer incubation times (up to 24 hours) has no influence on the result obtained.
Each experiment allows the measurement of only one sample. The latter is first incubated at an initial temperature of 20xc2x0 C., heated to 80xc2x0 C. over 38 minutes, left for 5 minutes at 80xc2x0 C. and then cooled to 20xc2x0 C. over 62 minutes. During this time, the fluorescence is measured simultaneously at two emission wavelengths (515 nm and 588 nm) using 470 nm as excitation wavelength. A measurement is carried out every 30 seconds. The temperature of the water bath is recorded in parallel, and the fluorescence profile as a function of the temperature is reconstituted from these values. The fluorescence profiles are then normalized between 20xc2x0 C. and 80xc2x0 C., and the temperature for which the intensity of emission at 515 nm is the mean of those at high and low temperature is called Tm. Under these conditions, the Tm of the reference sample without addition of product is 44xc2x0 C. in a lithium chloride buffer. This temperature is increased to more than 55xc2x0 C. in a sodium chloride buffer. The addition of a G-quadruplex-stabilizing compound induces an increase in the Tm. This increase is judged to be significant if it is greater than 3xc2x0.
The antitelomerase biological activity is determined by the following experimental protocol:
The leukaemia line HL60 is obtained from ATCC (American Type Culture Collection, Rockville, USA). The cells are cultured in suspension in RPMI 1640 medium containing L-glutamine at 2 mM, penicillin 200 U/ml, streptomycin 200 xcexcg/ml, gentamycin 50 xcexcg/ml and supplemented with 10% heat-inactivated foetal calf serum.
An aliquot of 105 cells is centrifuged at 3000xc3x97G and the supernatant discarded. The cell pellet is resuspended by several successive pipettings in 200 xcexcl of lysis buffer containing 0.5% CHAPS, 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM xcex2-mercaptoethanol, 0.1 mM PMSF and 10% glycerol and is stored in ice for 30 minutes. The lysate is centrifuged at 160,000xc3x97G for 20 minutes at 4xc2x0 C. and 160 xcexcl of supernatant are recovered. The proteins in the extract are assayed by the Bradford method. The extract is stored at xe2x88x9280xc2x0 C.
The inhibition of the telomerase activity is determined by a protocol for extension of the oligonucleotide TS (5xe2x80x2 AATCGTTCGAGCAGAGTT3xe2x80x2), in the presence of a cellular extract enriched in telomerase activity and compounds which are added at various concentrations (10, 1, 0.1 and 0.01 xcexcg/ml). The extension reaction is followed by a PCR amplification of the extension products with the aid of the oligonucleotides TS and CXext (5xe2x80x2 GTGCCCTTACCCTTACCCTTACCCTAA3xe2x80x2).
The reaction medium is prepared based on the following composition:
Double-distilled water QS . . . 50 xcexcl
The oligonucleotides are obtained from Eurogentec (Belgium) and are stored at xe2x88x9220xc2x0 C. at a stock concentration of 1 mg/ml in distilled water.
The reaction samples are assembled in 0.2 ml PCR tubes and one drop of paraffin oil is deposited on each of the reactions of the experiment before closing the tubes.
The reaction samples are then incubated in a Cetus 4800-type PCR apparatus under the following temperature conditions:
15 minutes at 30xc2x0 C.,
1 minute at 90xc2x0 C.,
followed by 30 cycles of,
30 seconds at 94xc2x0 C.,
30 seconds at 50xc2x0 C.,
and 1 minute 30 seconds at 72xc2x0 C.,
followed by a final cycle of 2 minutes at 72xc2x0 C.
For each of the samples, an aliquot of 10 xcexcl is pipetted under the oil layer and mixed with 5 xcexcl of a loading buffer containing:
The samples are then analysed by electrophoresis on 12% acrylamide gel in a 1xc3x97TBE buffer for 1 hour at a voltage of 200 volts, with the aid of a Novex electrophoresis system.
The acrylamide gels are then dried on a sheet of whatmann 3MM paper at 80xc2x0 C. for 1 hour.
The analysis and the quantification of the reaction products are carried out with the aid of an InstantImager apparatus (Pacard).
For each compound concentration tested, the results are expressed as percentage inhibition of the reaction and calculated from the untreated enzymatic control and from the enzyme-free sample (blank) according to the following formula:
(compound valuexe2x88x92blank value/enzymatic control valuexe2x88x92blank value)xc3x97100.
The concentration of compound inducing a 50% inhibition of the telomerase reaction (IC50) is determined with the aid of a semilogarithmic graphical representation of the inhibition values obtained as a function of each of the compound concentrations tested.
A compound is considered to be active as an antitelomerase agent when the quantity inhibiting 50% of the telomerase reaction is in particular less than 5 xcexcM.
The human cell lines KB and A549 are obtained from ATCC (American Type Culture Collection, Rockville, USA). The A549 cells are cultured in a layer in a culture flask in RPMI 1640 medium containing L-glutamine at 2 mM, penicillin 200 U/ml, streptomycin 200 xcexcg/ml and supplemented with 10% heat-inactivated foetal calf serum. The KB cells are cultured in a layer in a culture flask in Dulbelco""s medium containing L-glutamine at 2 mM, penicillin 200 U/ml, streptomycin 200 xcexcg/ml and supplemented with 10% heat-inactivated foetal calf serum.
The cells at the exponential growth phase are trypsinized, washed in 1xc3x97PBS and are inoculated in 96-well microplates (Costar) in an amount of 4xc3x97104 cells/ml for A549 and of 1.5xc3x97104 cells/ml (0.2 ml/well) and then incubated for 96 hours in the presence of variable concentrations of product to be studied (10, 1, 0.1 and 0.01 xcexcg/ml, each point in quadruplicate). 16 hours before the end of the incubation, 0.02% final of neutral red is added to each well. At the end of the incubation, the cells are washed with 1xc3x97PBS and lysed with 1% sodium lauryl sulphate. The cellular incorporation of the dye, which reflects cellular growth, is evaluated by spectrophotometry at a wavelength of 540 nm for each sample with the aid of a Dynatech MR5000 reading apparatus.
For each compound concentration tested, the results are expressed as percentage inhibition of cellular growth and calculated from the untreated control and the culture medium free of cells (blank) according to the following formula:
(compound valuexe2x88x92blank value/cell control valuexe2x88x92blank value)xc3x97100.
The concentration of compound inducing a 50% inhibition of growth (IC50) is determined with the aid of a semilogarithmic graphical representation of the inhibition values obtained as a function of each of the compound concentrations tested.
A compound is considered to be active as cytotoxic agent if the concentration inhibiting the growth of the tumour cells tested by 50% is in particular less than 10 xcexcM.